TY - JOUR A1 - Brendtke, Rico A1 - Wiehl, Michael A1 - Groeber, Florian A1 - Schwarz, Thomas A1 - Walles, Heike A1 - Hansmann, Jan T1 - Feasibility Study on a Microwave-Based Sensor for Measuring Hydration Level Using Human Skin Models JF - PLoS ONE N2 - Tissue dehydration results in three major types of exsiccosis—hyper-, hypo-, or isonatraemia. All three types entail alterations of salt concentrations leading to impaired biochemical processes, and can finally cause severe morbidity. The aim of our study was to demonstrate the feasibility of a microwave-based sensor technology for the non-invasive measurement of the hydration status. Electromagnetic waves at high frequencies interact with molecules, especially water. Hence, if a sample contains free water molecules, this can be detected in a reflected microwave signal. To develop the sensor system, human three-dimensional skin equivalents were instituted as a standardized test platform mimicking reproducible exsiccosis scenarios. Therefore, skin equivalents with a specific hydration and density of matrix components were generated and microwave measurements were performed. Hydration-specific spectra allowed deriving the hydration state of the skin models. A further advantage of the skin equivalents was the characterization of the impact of distinct skin components on the measured signals to investigate mechanisms of signal generation. The results demonstrate the feasibility of a non-invasive microwave-based hydration sensor technology. The sensor bears potential to be integrated in a wearable medical device for personal health monitoring. KW - gels KW - microwave radiation KW - collagens KW - skin physiology KW - reflection KW - skin anatomy KW - epidermis KW - antennas Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-179934 VL - 11 IS - 4 ER - TY - THES A1 - Groeber, Florian T1 - Etablierung eines vaskularisierten Hautäquivalentes T1 - Establishment of a vascularized skin equivalent N2 - Durch Methoden des Tissue Engineerings hergestellte dreidimensionale Hautäquivalente bilden die native humane Haut hinsichtlich ihrer histologischen Architektur, zellulären Zusammensetzung und metabolischen Aktivität ab. Diese Gewebe eignen sich daher als zellbasierte Wundauflagen für großflächige Hautdefekte oder als In-vitro-Testsysteme für den Ersatz von Tierversuchen. Bei bisherigen Hautäquivalenten fehlt jedoch ein funktionelles Blutgefäßsystem. Wird solch ein Gewebe als Implantat eingesetzt, führt das Fehlen von Blutgefäßen zu einer unzureichenden Versorgung mit Nährstoffen und zur Nekrose. Neben dieser klinischen Limitation ist auch das Anwendungsspektrum als In-vitro-Testsystem begrenzt. Bei nicht vaskularisierten Hautmodellen kann eine transdermale Penetration von Substanzen nicht akkurat abgeschätzt werden, da die zusätzliche Barriere, welche die gefäßauskleidenden Endothelzellen bilden, nicht enthalten ist. In Studien zur Integration eines Gefäßsystems in Hautäquivalente konnte bislang lediglich gezeigt werden, dass sich Endothelzellen zu gefäßartigen Strukturen zusammenlagern. Die Bildung von funktionellen perfundierbaren Gefäßen in einem in vitro generierten Hautäquivalent ist bisher jedoch noch nicht belegt. Entsprechend ist eine direkte Anastomose mit dem Blutkreislauf eines Patienten bei einem klinischen Einsatz als Hautimplantat nicht möglich. Bei einer Anwendung in In-vitro-Studien ist zudem das Gefäßsystem experimentell nicht zugänglich. In der vorliegenden Arbeit kann durch die Kombination einer biologischen, vaskularisierten Trägerstruktur (BioVaSc) mit einem neu entwickelten Bioreaktorsystems, ein Hautäquivalent mit einem perfundierbaren Gefäßsystem hergestellt werden. Die Generierung dieser sogenannten SkinVaSc erfolgt über die Besiedlung der BioVaSc mit humanen Keratinozyten (hEK) und Fibroblasten. Parallel dazu werden die eingebetteten Gefäßstrukturen der BioVaSc mit humanen mikrovaskulären Endothelzellen (hDMEC) rebesiedelt. Durch eine Anastomose zwischen den Gefäßen der BioVaSc und dem Bioreaktorsystem ist eine Perfusion mit physiologisch, gepulsten Drücken zwischen 80 und 120 mmHG möglich. Optimale Kulturbedingungen für die Haut- zellen können ferner durch zwei Kulturmodi generiert werden. Zur optimalen Versorgung der hEK innerhalb einer Proliferationsphase, die sich an die Zellaussaat anschließt, erfolgt eine kontinuierliche Versorgung der Oberfläche der SkinVaSc mit Medium. Der zweite Modus stimuliert die Differenzierung der hEK durch eine Kultivierung des Modells an der Grenzfläche zwischen Luft und Medium. Nach einer vierzehntägigen Kultivierung der SkinVaSc an der Luft Medium Grenzfläche lässt sich die Bildung einer hautspezifischen histologischen Architektur durch Hämalaun/Eosin und immunhistologische Färbungen belegen. Eine natürlich differenzierte Epidermis wird durch eine Basalmembran, die Kollagen Typ IV und Laminin 5 enthält von einen dermalen Teil getrennt. Die Dermale-Epidermale-Verbindung erscheint durch die Mikrostrukturierung der BioVaSc wellenförmig. Damit bildet die SkinVaSc die papillare Struktur der nativen humanen Haut ab. Innerhalb des dermalen Anteils können zudem Gefäßstrukturen ausgemacht werden. Die Innenseite der Gefäße sind durch eine Schicht aus hDMEC ausgekleidet, die endothelzellspe- zifische Oberflächenmarker wie "platelet endothelial cell adhesion molecule 1“ und "von Willebrand Faktor“ aufweisen. Eine zerstörungsfreie Überwachung der SkinVaSc hinsichtlich der epidermalen Differenzierung ist durch eine integrierte Sensortechnologie auf Basis der Impedanz-spektroskopie möglich. Dabei erlaubt ein entwickeltes mathematisches Modell die Extraktion von biologisch relevanten Informationen aus Impedanzspektren in einem Frequenzbereich zwischen 1 Hz und 100 kHz. Innerhalb dieser Studien ließ sich zeigen, dass die epidermale Differenzierung zu einer signifikanten Steigerung des ohmschen Widerstandes von 245,3 Ohm*cm2 zu 1108,1 Ohm*cm2 führt. Gleichzeitig sinkt die zelluläre Kapazität von 131,5µF/cm2 auf 5,4µF/cm2 ab. Durch diese Parameter ist es möglich die epidermale Barriere zerstörungsfrei über die Kultivierungszeit zu überwachen. Das Gefäßsystem der SkinVaSc ermöglicht es mehr dermatologische Fragestellungen in vitro zu untersuchen und damit Tierversuche zu ersetzen. Zudem kann auf Basis der SkinVaSc ein vaskularisiertes Hautimplantat entwickelt werden, das es ermöglicht tiefe Hautverletzungen zu behandeln. N2 - Tissue engineered three-dimensional skin equivalents can mimic the key anatomical, metabolic and cellular aspects of the human skin and thus can be employed as wound coverage for large skin defects or as in vitro test systems as an alternative to animal models. However, current skin equivalents lack a functional vasculature. Hence, their possible applications are limited in both fields. In a clinical application, the absence of a vasculature can lead to an insufficient supply of nutrients, which is a major reason for the failure of skin grafts. Moreover, without a vascular system, skin equivalents are not suitable to assess the transdermal penetration of substances accurately as the additional barrier of the endothelial cells is not present. Although there are approaches where endothelial cells are seeded into the dermal part of full thickness skin equivalents, which yield alignment of endothelial cells to vessel like structures, no functional perfusable vasculature is formed in vitro. Thus, no direct anastomosis of a skin graft with a patient’s blood flow is possible and the vasculature is not experimentally accessible in in vitro tests. Using a biological vascularized scaffold (BioVaSc) in combination with a custom developed bioreactor system, this thesis documents the in vitro generation of a vas- cularized skin equivalent with a perfused vascular network. The BioVaSc is based on a decellularized segment of a porcine jejunum and consists mainly of a collagen type III and I scaffold, in which the structure of the former vascular network is still embedded. For the formation of a vascularized skin equivalent (termed ‘Skin- VaSc’ in this thesis), the BioVaSc is initially constructed with human fibroblasts and keratinocytes. Following this, the embedded vascular structures in the BioVaSc are seeded with human micro vascular endothelial cells (hDMEC). This hDMEC vasculature in the BioVaSc is then connected to an outer fluidic system, which is provided by a developed bioreactor system. The fluidic system generates a physio- logical medium flow into the BioVaSc with a pulsatile pressure profile between 80 and 120 mmHg and can culture the SkinVaSc both under submersed conditions and at the air-liquid-interface. After culturing the SkinVaSc at the air-liquid interface for 14 days, histological hemalaun-eosin and immunohistological staining revealed specific histological architecture representative of the human dermis in vivo. A naturally differentiated epidermal layer and a dermal equivalent are separated by a basement membrane with components such as collagen type IV and laminin 5. Due to the villi structure of the BioVaSc, the dermal-epidermal-junction exhibits a papillary like architecture as seen in human skin in vivo. Additionally, hDMEC are detectable inside the per- fused vasculature and exhibit endothelial cell specific surface markers such as von Willebrand Factor (vWF) and platelet endothelial cell adhesion molecule 1. To monitor the formation of skin tissue inside the bioreactor, a non-destructive sen- sor technology based on impedance spectroscopy was established. A derived algorithm allows one to extract biologically relevant information from impedance spectra between 1 Hz and 100 kHz. Employing this algorithm, a drop from 131.5µF/cm2 to 5.4To monitor the formation of skin tissue inside the bioreactor, a non-destructive sensor technology based on impedance spectroscopy was established. A derived algo rithm allows one to extract biologically relevant information from impedance spectra between 1 Hz and 100 kHz. Employing this algorithm, a drop from 131.5µF/cm2 to 5.4µF/cm2 of the capacity and an increase from 245.3 Ohm*cm2 to 1108.1 Ohm*cm2 of the resistance was detectable from day 1 to day 12 of the culture at the air-liquid-interface. These changes could be correlated to the differentiation of the keratinocytes and the formation of a corneous layer. Thus, this method can be used to assess the epidermal differentiation non-invasively. Due to the integrated vasculature the SkinVaSc enables one to investigate additional dermatological questions in vitro and thus to replace animal experiments. Moreover, the SkinVaSc has enormous potential to be used as a vascularized skin graft for the treatment of deep skin wounds. µF/cm2 of the capacity and an increase from 245.3 Ohm*cm2 to 1108.1 Ohm*cm2 of the resistance was detectable from day 1 to day 12 of the culture at the air-liquid- interface. These changes could be correlated to the differentiation of the keratinocy- tes and the formation of a corneous layer. Thus, this method can be used to assess the epidermal differentiation non-invasively. Due to the integrated vasculature the SkinVaSc enables one to investigate additional dermatological questions in vitro and thus to replace animal experiments. Moreover, the SkinVaSc has enormous potential to be used as a vascularized skin graft for the treatment of deep skin wounds. KW - skin KW - vascularization KW - Bioengineering KW - Alternative methods KW - Vaskularisierung KW - Alternativmethoden KW - Tisuue Engineering KW - Haut KW - Vaskularisation Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-107453 ER - TY - JOUR A1 - Groeber, Florian A1 - Engelhardt, Lisa A1 - Lange, Julia A1 - Kurdyn, Szymon A1 - Schmid, Freia F. A1 - Rücker, Christoph A1 - Mielke, Stephan A1 - Walles, Heike A1 - Hansmann, Jan T1 - A First Vascularized Skin Equivalent as an Alternative to Animal Experimentation JF - ALTEX - Alternatives to Animal Experimentation N2 - Tissue-engineered skin equivalents mimic key aspects of the human skin, and can thus be employed as wound coverage for large skin defects or as in vitro test systems as an alternative to animal models. However, current skin equivalents lack a functional vasculature limiting clinical and research applications. This study demonstrates the generation of a vascularized skin equivalent with a perfused vascular network by combining a biological vascularized scaffold (BioVaSc) based on a decellularized segment of a porcine jejunum and a tailored bioreactor system. Briefly, the BioVaSc was seeded with human fibroblasts, keratinocytes, and human microvascular endothelial cells. After 14 days at the air-liquid interface, hematoxylin & eosin and immunohistological staining revealed a specific histological architecture representative of the human dermis and epidermis including a papillary-like architecture at the dermal-epidermal-junction. The formation of the skin barrier was measured non-destructively using impedance spectroscopy. Additionally, endothelial cells lined the walls of the formed vessels that could be perfused with a physiological volume flow. Due to the presence of a complex in-vivo-like vasculature, the here shown skin equivalent has the potential for skin grafting and represents a sophisticated in vitro model for dermatological research. KW - alternative to animal testing KW - skin equivalents KW - tissue engineering KW - vascularization Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164438 VL - 33 IS - 4 ER - TY - JOUR A1 - Groeber, Florian A1 - Schober, Lena A1 - Schmid, Freia F. A1 - Traube, Andrea A1 - Kolbus-Hernandez, Silvia A1 - Daton, Karolina A1 - Hoffmann, Sebastian A1 - Petersohn, Dirk A1 - Schaefer-Korting, Monika A1 - Walles, Heike A1 - Mewes, Karsten R. T1 - Catch-up validation study of an in vitro skin irritation test method based on an open source reconstructed epidermis (phase II) JF - Toxicology in Vitro N2 - To replace the Draize skin irritation assay (OECD guideline 404) several test methods based on reconstructed human epidermis (RHE) have been developed and were adopted in the OECD test guideline 439. However, all validated test methods in the guideline are linked to RHE provided by only three companies. Thus,the availability of these test models is dependent on the commercial interest of the producer. To overcome this limitation and thus to increase the accessibility of in vitro skin irritation testing, an open source reconstructed epidermis (OS-REp) was introduced. To demonstrate the capacity of the OS-REp in regulatory risk assessment, a catch-up-validation study was performed. The participating laboratories used in-house generated OS-REp to assess the set of 20 reference substances according to the performance standards amending the OECD test guideline 439. Testing was performed under blinded conditions. The within-laboratory reproducibility of 87% and the inter-laboratory reproducibility of 85% prove a high reliability of irritancy testing using the OS-REp protocol. In addition, the prediction capacity was with an accuracy of 80% comparable to previous published RHE based test protocols. Taken together the results indicate that the OS-REp test method can be used as a standalone alternative skin irritation test replacing the OECD test guideline 404. KW - Model KW - Chemicals KW - Assay KW - Episkin KW - Vivo KW - RHE KW - In vitro skin irritation testing KW - Open source reconstructed epidermis KW - Validation KW - Alternative test methods Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-187311 VL - 36 ER -